This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The vacuolar ATPase (V-ATPase) is a multi subunit rotary motor enzyme that functions as an ATP hydrolysis driven proton pump in the endomembrane system of eukaryotic cells. The V-ATPase consistes of two motor domains, a cytoplasmic ATPase (V1) and a membrane bound proton channel (V0). The two domains are linked by three peripheral stator proteins that function as a structural link to counteract the rotational torque that is generated during ATP hydrolysis. We have recently obtained a 3-D reconstruction of the intact V-ATPase from yeast (Zhang et al., JBC 283, 35983) and we are now using X-ray crystallography to determine the atomic resolution structures of V-ATPase subunits and subunit domains for fitting into the EM derived map. We have crystallized the peripheral stalk forming subunits of yeast V-ATPase (subunits E&G) in complex with a domain of subunit C. A preliminary diffraction analysis performed at the Chess beamline F1 (Fall 2009;in collaboration with Dr. Edward Berry) resulted in ~5.5 [unreadable] diffraction. The crystals belong to spacegroup P212121 with unit cell parameters of 95.2, 114.1, 133.9 [unreadable] , 90,90.5,90[unreadable][unreadable]. Currently, there is no crystal structure available for the peripheral stalk(s) of the V-ATPase (or any of the related rotary ATPases including the F1F0-ATP synthase or the archaeal A-ATPase). SInce last fall, we have optimized crystallization conditions including a 96 condition additive screen, leading to numerous conditions with varying crystal morphologies. We will use the beam time at Chess, if approved, to screen crystals for high quality diffraction and to collect native data sets if time and crystal quality permits. Subsequent work, for which a full proposal is planned, would include heavy metal soaks and/or SeMet containing protein. Molecular replacement may be possible as a crystal structure for subunit C is available.